1. Field of the Invention
The purpose of this invention is to assay the quantity and quality of Aβ peptide in Alzheimer's disease (AD) and Aβ amyloidotic disorders related to Alzheimer's disease. Specifically, the invention proposes to achieve this end by enriching the peptide by capturing it from biological fluids such as plasma, serum, cerebrospinal fluid or urine with a zinc- or copper-chelated microwell plate, and then measuring the amounts of captured Aβ with specific anti-Aβ antibodies in an ELISA.
2. Related Art
Alzheimer's disease is characterized pathologically by the accumulation in the brain of Aβ protein. The Aβ protein is a small peptide that is also found cerebrospinal fluid and plasma. Much evidence implicates the accumulation of Aβ in the pathogenesis of the disease, either as the neurotoxic agent itself or as a hallmark which accompanies neurotoxicity in the disorder. Aβ accumulates as a highly insoluble deposit within neuronal tissues. It is desirable to discover a treatment which would reverse the deposition and relieve or arrest clinical deterioration.
Aggregation of Aβ in the brain is believed to contribute to the progressive dementia, characteristic of Alzheimer's disease (AD) and to the premature AD observed among Down's syndrome patients. Aβ, a 4.3-kDa peptide, is the principal constituent of the cerebral amyloid deposits, a pathological hallmark of Alzheimer's disease (AD) (Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985); Glenner & Wong, Biochem. Biophys. Rev. Commun. 120:885-890 (1984)). Aβ is derived from the much larger amyloid protein precursor (APP) (Kang et al., Nature 325:733-736 (1987); Tanzi et al., Science 235:880-884 (1987); Robakis et al., Proc. Natl. Acad. Sci. USA 84:4190-4194 (1987); Goldgaber et al., Science 235:877-880 (1987)), whose physiological function remains unclear. The cause of Alzheimer's disease remains elusive; however, the discovery of mutations of APP close to or within the Aβ domain (Goate et al., Nature 349:704-706 (1991); Levy et al., Science 248:1124-1126 (1990); Murrell et al., Science 254:97-99 (1991); Hendricks et al., Nature Genet. 1:218-221 (1992), linked to familial AD (FAD) (E. Levy et al., Science 248:1124 (1990); Aβ Goate et al., Nature 349:704 (1991); M. Chartier-Harlin et al., Nature 353:844 (1991); J. Murrell, M. Farlow, B. Ghetti, M. D. Benson, Science 254:97 (1991); L. Hendricks et al., Nature Genet. 1:218 (1992); M. Mullan et al., Nature Genet. 1:345 (1992)), indicates that the metabolism of Aβ and APP is likely to be intimately involved with the pathophysiology of this disorder.
Alzheimer's disease affects 10% of individuals over the age of 60, however, the existence of Aβ deposits in 40% of the brains of normal individuals in their forties suggests an even larger subclinical prevalence. Hence, the disease process is likely to be very common, with individual thresholds of neuronal and functional reserve being responsible for the varying onset of clinical symptoms. The disease is debilitating, chronic, incurable and very expensive to treat and an effective prevention or therapy would have an enormous commercial market. However, there are no reliable biochemical markers for AD.
FAD patients with the “Swedish” APP mutation overproduce the soluble, secreted form of Aβ and suffer from early onset (<60 years) AD (Citron et al., Nature 360:672-674 (1992)). A potential neuropathogenic mechanism has been reported by Younkin and colleagues (Society for Neuroscience, Mol. Genet. Med. 3:95-137 (1993)) for the APP 717 mutations which account for 90% of the APP mutations causing FAD. These mutations apparently lead to an increase in the ratio of “long” Aβ (1-42) to Aβ (1-40). Aβ (1-40) is the predominant species in the cerebrospinal fluid (CSF), and is a relatively soluble peptide. Aβ (1-42) is significantly more amyloidogenic, and its overproduction relative to the 1-40 species appears to lead to early-onset AD in these patients. Therefore, levels of Aβ (1-40) and Aβ (1-42) in the cerebrospinal fluid (CSF), plasma, serum or urine may be expected to correlate with cerebral pathology in sporadic AD cases, the predominant clinical form of the disorder.
Two protocols currently exist for the estimation of Aβ levels in biological fluids. The first involves the immunoprecipitation of Aβ with specific anti-Aβ antibodies (e.g. Haass et al., Nature 359:322-325 (1992); Citron et al., Nature 360:672-674 (1992)), a technique which is, at best, semiquantitative. This technique was used in combination with western blotting to measure Aβ levels in CSF (Shoji et al., Science 258:126-129 (1992)) but found no gross differences between AD and control specimens. A double antibody capture ELISA using monoclonal antibodies raised against Aβ appears to give specific Aβ quantification with a sensitivity limit at about 0.6 nM (Seubert et al., Nature 359:325-327 (1992)).
The double antibody ELISA is more widely used and is the only described means of accurately quantifying Aβ. It has two important limitations. It requires an abundance of expensive antibody to coat the wells of microwell plates in order to capture the Aβ from the biological fluid. A second anti-Aβ antibody, at a higher dilution, is used to detect captured Aβ. The second limitation of the double-antibody capture ELISA technique for Aβ assay is that it requires a fluorescence-generating enzyme-conjugated detection antibody and a fluorescence microwell plate reader for the final step of the assay.
Fluorescence plate readers are highly specialized and expensive (about $30,000, Millipore Cytofluor), which limits the accessibility of the technique. Fluorescence has been preferred over more versatile, and cheaper, chromogenic assays (e.g., horse radish peroxidase-conjugated detection antibody acting on a chromogenic substrate), because it lowers the limit of sensitivity allowing the measurement of Aβ at the levels found in biological fluids. No Aβ assay has been described where the development of a chromogenic substrate was measured by a visible-light microwell plate reader, a far less expensive instrument (e.g., $8,000).
The Aβ species assay of the present invention will provide a rational basis to monitor response to putative treatments for AD, as well as providing early diagnostic information if clinical outcome studies validate the correlation of the Aβ levels in the blood or CSF with disease progression.